Ultrastructural assessment of medically relevant levels of synthesized nanosilver-exposed canine blood mononuclear leukocytes reveals irreversible immunocytotoxicity.

This study investigated the cytotoxic effects of AgNPs on canine PBMCs at different nominal concentrations and exposure times using cytometric and ultrastructural assessments. AgNPs have been studied extensively for biomedical applications; however, the conditions under which immune cells shift from regulated cell death to uncontrolled cell damage are still not fully understood. In this study, canine PBMCs were exposed to green-synthesized AgNPs at increasing nominal concentrations (20, 50, and 100 μg/mL) under short-term (24 h) and prolonged (96 h) exposure conditions. Annexin V/PI staining distributions were quantitatively assessed using flow cytometry, while ultrastructural alterations were descriptively examined by scanning and transmission electron microscopy. The results revealed a clear concentration- and time-dependent shift in PBMC responses. The results showed progressive changes in PBMC viability and cell-death profiles with increasing AgNP concentration and exposure time. At 20 μg/mL, cell viability decreased from 59.47 ± 6.22% at 24 h to 1.58 ± 0.71% at 96 h. At higher AgNP concentrations, viable-cell percentages decreased further, accompanied by increased proportions of Annexin V+/PI+ and Annexin V-/PI+ cells. These cytometric findings were accompanied by descriptive ultrastructural observations of membrane alterations, organelle disruption, and loss of cellular integrity in the examined images. Overall, this study provides integrated cytometric data and descriptive ultrastructural observations of concentration- and time-associated shifts in cell-death staining and morphological patterns in AgNP-exposed canine PBMCs. These findings highlight the importance of considering AgNP concentration and exposure duration when evaluating their biological effects under the tested in vitro conditions.